Abstract
Targeting MAPK pathway using a combination of BRAF and MEK inhibitors is an efficient strategy to treat melanoma harboring BRAF-mutation. The development of acquired resistance is inevitable due to the signaling pathway rewiring. Combining western blotting, immunohistochemistry, and reverse phase protein array (RPPA), we aim to understanding the role of the mTORC1 signaling pathway, a center node of intracellular signaling network, in mediating drug resistance of BRAF-mutant melanoma to the combination of BRAF inhibitor (BRAFi) and MEK inhibitor (MEKi) therapy. The mTORC1 signaling pathway is initially suppressed by BRAFi and MEKi combination in melanoma but rebounds overtime after tumors acquire resistance to the combination therapy (CR) as assayed in cultured cells and PDX models. In vitro experiments showed that a subset of CR melanoma cells was sensitive to mTORC1 inhibition. The mTOR inhibitors, rapamycin and NVP-BEZ235, induced cell cycle arrest and apoptosis in CR cell lines. As a proof-of-principle, we demonstrated that rapamycin and NVP-BEZ235 treatment reduced tumor growth in CR xenograft models. Mechanistically, AKT or ERK contributes to the activation of mTORC1 in CR cells, depending on PTEN status of these cells. Our study reveals that mTOR activation is essential for drug resistance of melanoma to MAPK inhibitors, and provides insight into the rewiring of the signaling networks in CR melanoma.
Highlights
Metastatic melanoma is the most aggressive form of skin cancer [1]
RESULTS mTOR complex 1 (mTORC1) activity is restored in melanoma cells resistant to BRAF inhibitor (BRAFi)/MEK inhibitor (MEKi) combination To better understand the effect of combined BRAFi and MEKi on resistance-associated mTORC1 signaling, we treated a panel of paired parental and combination therapy (CR) BRAF mutant melanoma cell lines with PLX4720 (PLX, 2.5 μM, BRAFi) and PD0325901 (PD, 0.25 μM, MEKi) for 24 h (Fig. 1)
To test our hypothesis that mTORC1 activity was initially suppressed during short-term MAPKi and rebounded after long-term treatment, we examined 1205Lu xenografts and WM3929 patient-derived xenograft (PDX) tumors that were untreated, short-term treated with BRAF and MEK inhibitors, or a long-term treated until resistance arose
Summary
Metastatic melanoma is the most aggressive form of skin cancer [1]. Approximately 50% of cutaneous melanomas harbor activating BRAF mutations, which drive hyperactivation of the MitogenActivated Protein Kinase (MAPK) signaling pathway [2, 3]. Targeting MAPK pathway using a combination of BRAF and MEK inhibitors elicits a 70% response rate in patients with BRAF-mutant melanoma [4,5,6]. Molecular mechanisms that underlie acquired resistance to BRAF inhibitor monotherapy can be attributed to the reactivation of ERK, activating mutations in NRAS, alternative activation of RTK-mediated pathways, amplification or truncation of BRAF, overexpression of COT, mutations in MEK1 and other genetic events [5,6,7,8,9,10]. In terms of resistance to BRAFi and MEKi combination therapy (CR), several mechanisms including the development of MEK2 mutations, acquisition of concurrent BRAF/NRAS mutations, amplification of BRAF, ER translocation of the ERK and others have been reported [11,12,13,14,15,16]. MAPK-independent mechanisms such as the rewiring of RAC1/CDC42-PAK signaling pathway [17], alternative activation of the PI3K/AKT signaling axis [15], and immune-related components in tumor microenvironments contribute to CR [18]
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